新型接合型NDM-1质粒及SGI1/PGI1在介导XDR肠杆菌科细菌产生中的作用

The sporadic emergence of New Delhi metallo-β-lactamase 1 (NDM-1) producing Enterobacteriaceae in mainland China has been reported increasingly since 2013. Unexpectedly, the prevalence and active spread of blaNDM-1 in Enterobacteriaceae was observed in Henan province, leading to the emergence of extensively drug-resistant (XDR) isolates, presents an urgent threat to human health.Given that the blaNDM-1 gene is located mostly on conjugative plasmids and always associated with other antibiotic resistance determinants, the acquisition of conjugative plasmids co-harboring blaNDM-1 and multiple resistance genes in clinical Enterobacteriaceae isolates may lead to the emergence of XDR strains and seriously limit the therapeutic options. Our recent study demonstrated that a pan-susceptible Escherichia coli J53 became an XDR strain susceptible only to tigecycline and colistin after accepting a self-transferable NDM-1 plasmid from a clinical XDR isolate of Enterobacter cloacae via conjugation under the laboratory conditions. This self-transferable plasmid of ca.360 kb is unique, which co-harboring multiple resistance determinants including blaNDM-1, fosA3 and armA, and may belong to a novel incompatibility group. In order to characterize the novel conjugative plasmid, the whole sequence analysis and fitness cost assay will be conducted to illuminate the structure, evolution and capability for dissemination of resistance genes of this plasmid. Moreover, a novel variant of Salmonella genomic island 1 (SGI1-Z) carrying multiple resistance genes was identified in an XDR Proteus mirabilis which harbored a similar conjugative NDM-1 plasmid, this finding together with a recent report of the emergence of a novel SGI1 variant, named PGI1 (Proteus genomic island 1) carrying blaNDM-1 in a P. mirabilis clinical isolate, indicating the integrative mobilizable elements of SGI1/PGI1 may play an important role in dissemination resistance genes including blaNDM-1. Thus, we plan to evaluate the prevalence, molecular characterization and mobility of SGI1/PGI1 in carbapenem-resistant P. mirabilis to highlight the potential role of these elements in spread of resistance determinants.

近期，NDM-1酶在河南地区肠杆菌科细菌中出现高度流行和跨种属传播，导致了不同种属XDR菌株的产生，给临床治疗带来了极大困难。质粒是介导blaNDM-1播散的重要工具。我们前期在1株XDR 阴沟肠杆菌中发现了一个不相容性未知的新型NDM-1质粒，该质粒可通过接合转移使全敏感的E. coli J53变成XDR菌株。另外，1株XDR奇异变形杆菌在携带类似质粒的同时还携带沙门氏菌基因岛1的新变异体（SGI1-Z），结合最新报道的携带blaNDM-1的奇异变形杆菌基因岛1（PGI1），我们推测结构多变的可移动元件SGI1/PGI1将在介导blaNDM-1传播中发挥重要作用。本项目拟通过全质粒测序分析和适应性代价研究探讨新型NDM质粒的进化过程和播散稳定性；同时探查SGI1/PGI1在奇异变形杆菌中的流行，通过结构解析和可转移性分析，探讨该类元件在介导blaNDM-1传播和XDR菌株产生中的作用。

产NDM酶肠杆菌科细菌耐药谱广、治疗困难，感染患者病死率高，给临床抗感染治疗带来了严重威胁。本项目前期发现NDM-1酶在河南地区分离的肠杆菌科细菌中出现高发流行，并导致了XDR菌株的产生。项目以此为切入点，解析了介导NDM传播的移动元件（质粒及转座元件等）的分子特征；同时，在全基因组测序的基础上揭示XDR菌株形成的分子机制。初步探明了NDM酶区域性传播流行的规律，建立了有效检测分析方法。为防控NDM酶的进一步播散提供了有效数据和理论支持。项目重要结论如下：1、在NDM传播机制方面，通过持续研究发现（2011-2016年）从2013年起河南地区开始出现不同NDM亚型，其中NDM-5亚型从2014年起开始逐渐取代NDM-1成为主要流行亚型，在此研究过程中，我们发现并解析了国内首例产NDM-4酶大肠埃希菌（E. coli）ST410菌株；发现了介导NDM-5基因传播的新型接合型F2: A-: B-质粒，并推测该质粒可能是由国外常见的介导NDM传播的pMC-NDM-like质粒与国内报道的pSJ_94-like质粒重组产生；发现IncX3型质粒逐渐取代之前的IncA/C型质粒成为携带NDM传播的最主要载体，该质粒同时也能介导OXA-181基因的传播；发现了携带NDM酶的E. coli 以ST167型为主。2、在XDR菌株形成的分子机制方面，发现近期报道的多粘菌素耐药基因mcr-1的传播是导致产NDM酶E. coli成为XDR菌株的重要原因，基于全基因组测序，解析了NDM阳性菌株携带的新型杂合型mcr-1质粒，揭示了mcr-1基因在传播进化过程中的新特点。